The ability to faithfully replicate DNA is essential to all living cells. Before replication can ensue, replisomal machineries must first be properly constructed by specific replication initiation factors. Extensive studies have identified many of the proteins responsible for replication initiation and have produced a general framework for their action. Nonetheless, significant gaps remain in our understanding of this process, particularly, with respect to initiation protein mechanisms and the degree to which their function is conserved across the three domains of life. The long-term objective of this proposal is to illuminate several key structure/function relationships of the origin-binding initiator proteins, replicative helicases, and helicase-loader factors that mediate replisome assembly. To compare and contrast the function of these proteins in different organisms, we will study archaeal and bacterial replication initiation systems. Specifically, we aim to: 1) biochemically and structurally determine how the archaeal Cdc6/Orc1 protein interacts with specific replication origin sites, 2) reconstitute and biophysically characterize dimeric and trimeric initiator complexes on origin DNAs, and 3) determine the structure of a cellular, hexameric replicative helicase, complexed either with substrates such as ATP, or with a specialized loading factor. We have already pioneered structural studies of several bacterial and archaeal initiation proteins. To enable our new proposed efforts, we have: 1) expressed and purified over 70 different full-length and truncated initiation factors from six different organisms, 2) begun to define the interaction of several of these proteins with each other and DNA, and 3) obtained diffraction data and crystal forms for some of these factors. Information from these studies will broadly impact a number of important scientific research fronts, from understanding the assembly and function of molecular machines, to providing atomic resolution information on potential targets for new antimicrobials. Data obtained to date demonstrate the feasibility of our specific aims.